ROCK inhibition abolishes the establishment of the aquiferous system in Ephydatia muelleri (Porifera, Demospongiae)

Abstract Background Vitamin B3 (nicotinamide) plays important roles in metabolism as well as in SIRT and PARP pathways. It is also recently reported as a novel kinase inhibitor with multiple targets. Nicotinamide promotes pancreatic cell differentiation from human embryonic stem cells (hESCs). However, its molecular mechanism is still unclear. In order to understand the molecular mechanism involved in pancreatic cell fate determination, we analyzed the downstream pathways of nicotinamide in the derivation of NKX6.1+ pancreatic progenitors from hESCs. Methods We applied downstream modulators of nicotinamide during the induction from posterior foregut to pancreatic progenitors, including niacin, PARP inhibitor, SIRT inhibitor, CK1 inhibitor and ROCK inhibitor. The impact of those treatments was evaluated by quantitative real-time PCR, flow cytometry and immunostaining of pancreatic markers. Furthermore, CK1 isoforms were knocked down to validate CK1 function in the induction of pancreatic progenitors. Finally, RNA-seq was used to demonstrate pancreatic induction on the transcriptomic level. Results First, we demonstrated that nicotinamide promoted pancreatic progenitor differentiation in chemically defined conditions, but it did not act through either niacin-associated metabolism or the inhibition of PARP and SIRT pathways. In contrast, nicotinamide modulated differentiation through CK1 and ROCK inhibition. We demonstrated that CK1 inhibitors promoted the generation of PDX1/NKX6.1 double-positive pancreatic progenitor cells. shRNA knockdown revealed that the inhibition of CK1α and CK1ε promoted pancreatic progenitor differentiation. We then showed that nicotinamide also improved pancreatic progenitor differentiation through ROCK inhibition. Finally, RNA-seq data showed that CK1 and ROCK inhibition led to pancreatic gene expression, similar to nicotinamide treatment. Conclusions In this report, we revealed that nicotinamide promotes generation of pancreatic progenitors from hESCs through CK1 and ROCK inhibition. Furthermore, we discovered the novel role of CK1 in pancreatic cell fate determination.

Download Full-text

ROCK inhibition with Fasudil induces beta-catenin nuclear translocation and inhibits cell migration of MDA-MB 231 human breast cancer cells

Scientific Reports ◽

10.1038/s41598-017-14216-z ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 12

Author(s):

Fabiana Sélos Guerra ◽

Ramon Guerra de Oliveira ◽

Carlos Alberto Manssour Fraga ◽

Claudia dos Santos Mermelstein ◽

Patricia Dias Fernandes

Keyword(s):

Breast Cancer ◽

Cell Migration ◽

Cancer Cells ◽

Human Breast Cancer ◽

Breast Cancer Cells ◽

Nuclear Translocation ◽

Beta Catenin ◽

Human Breast Cancer Cells ◽

Human Breast ◽

Rock Inhibition

Download Full-text

ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression

Nucleic Acids Research ◽

10.1093/nar/gkv728 ◽

2015 ◽

Vol 43 (15) ◽

pp. 7577-7589 ◽

Cited By ~ 7

Author(s):

Takeshi Yoshikawa ◽

Jianfeng Wu ◽

Motoyuki Otsuka ◽

Takahiro Kishikawa ◽

Motoko Ohno ◽

...

Keyword(s):

Rock Inhibition ◽

Microrna Function

Download Full-text

ROCK inhibition stimulates SOX9/Smad3-dependent COL2A1 expression in inner meniscus cells

Journal of Orthopaedic Science ◽

10.1016/j.jos.2016.02.013 ◽

2016 ◽

Vol 21 (4) ◽

pp. 524-529 ◽

Cited By ~ 2

Author(s):

Takayuki Furumatsu ◽

Ami Maehara ◽

Toshifumi Ozaki

Keyword(s):

Col2a1 Expression ◽

Meniscus Cells ◽

Rock Inhibition

Download Full-text

ROCK Inhibition Facilitates In Vitro Expansion of Glioblastoma Stem-Like Cells

PLoS ONE ◽

10.1371/journal.pone.0132823 ◽

2015 ◽

Vol 10 (7) ◽

pp. e0132823 ◽

Cited By ~ 20

Author(s):

Samantha G. Tilson ◽

Elizabeth M. Haley ◽

Ursula L. Triantafillu ◽

David A. Dozier ◽

Catherine P. Langford ◽

...

Keyword(s):

In Vitro Expansion ◽

Rock Inhibition

Download Full-text

ROCK inhibition and CNTF interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells

Brain ◽

10.1093/brain/awm284 ◽

2007 ◽

Vol 131 (1) ◽

pp. 250-263 ◽

Cited By ~ 141

Author(s):

Paul Lingor ◽

Lars Tönges ◽

Nicole Pieper ◽

Christina Bermel ◽

Elisabeth Barski ◽

...

Keyword(s):

Retinal Ganglion Cells ◽

Ganglion Cells ◽

Signalling Pathways ◽

Retinal Ganglion ◽

Rock Inhibition

Download Full-text

Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

Blood ◽

10.1182/blood-2019-131947 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3607-3607

Author(s):

Grace Lee ◽

Sheela Karunanithi ◽

Zachary Jackson ◽

David Wald

Keyword(s):

Cell Therapy ◽

Cytotoxic Activity ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Akt Activation ◽

Nk Cell Cytotoxicity ◽

Rock Inhibition ◽

Nk Cell Therapy

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'>0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

Download Full-text